PROJECT SUMMARY/ABSTRACT Zika virus (ZIKV), an emerging arbovirus transmitted by the Aedes mosquitoes, has been correlated with newborn microcephaly and Guillain-Barre syndrome in adults. The World Health Organization has declared Zika a public health emergency and is calling for new diagnostics and enhanced surveillance. In particular, point-of-care (POC) diagnostics that enable rapid sharing of clinical and epidemiological data will facilitate international control and research efforts. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis of patient blood is the current standard for ZIKV diagnosis. Unfortunately, RT-PCR requires technical expertise, complex instruments, and infrastructure typically only available in clinical laboratories. Accurate, inexpensive diagnostic tools for patient care and epidemiological surveillance of insect vectors in remote or resource-limited areas are sorely lacking. To fill this critical need, we propose to develop a POC nucleic acid diagnostic that will identify ZIKV directly from mosquito vectors and from patient saliva that can not only be non-invasively sampled but is more frequently positive for ZIKV compared to blood. Paratus Diagnostics has previously design, developed and demonstrated a point-of-care system (the PreparedNOW system) that can meet the demanding challenges outlined above. As described in further detail below, the Paratus Diagnostics POC device has previously been shown to be capable of detecting ZIKV genes using a highly advanced and innovative molecular multiplexed assay developed by our partner, Prof. Andy Ellington of the University of Texas. Two variants of the basic Paratus PreparedNOW system have been demonstrated: one version is capable of processing actual mosquitos; the second version will accept human patient saliva as the specimen from which to perform a diagnostic test to determine if an individual has been infected with Zika. Our proposed system under this project will be capable of detecting biologically relevant concentrations of viral RNA in human saliva or infected mosquitoes within 60 minutes (Aim 2, Objective 4). Our platform leverages our patented and well-tested portfolio of devices; and several components have been reduced to practice and successfully tested. Our approach relies on a consumable cartridge for sample collection, preparation and molecular assay processing all within a housing that fits in the palm of a hand, and requires only a few intuitive user-operated steps. Development of this simple-but-robust approach to fast and accurate pathogen detection can be readily diversified to a wide array of pathogens and vectors. Our point-of-care diagnostic solution will be powered by the unique one-pot assay being developed by our partner Dr. Andrew Ellington (University of Texas, Austin). This assay incorporates several innovations. First, the unique amplification assay (RT-LAMPdeg) will be adapted for co-amplifying four ZIKV genes in a single assay (Aim 1, Objective 1). Second, false positives will be nearly eliminated by coupling the amplification assay with a unique nucleic acid probe (4GO) that will sequence-specifically transduce up to four ZIKV genes into single-channel fluorescence readable by the Paratus system (Aim 1, Objective 2). This will allow a single assay to accurately detect known ZIKV variants, fully accounting for most currently circulating Asian and African lineages. Third, fluorescence will be readout using our smartphone based system, and the assay will be proofed with clinical and biological samples (obtained from Dr. Grant Hughes, University of Texas Medical Branch) (Aim 2, Objective 3).